Actions of benzophenanthridine alkaloids and various synthetic compounds on the cannabinoid-1 (CB1) receptor pathway of mouse brain with particular reference to the effects on [3H]CP55940 and [3H]SR141716A binding, interference with basal and CP55940-stim

Benzophenanthridine alkaloids chelerythrine and sanguinarine inhibited the binding of 3HCP55940 and 3HSR141716A to mouse brain membranes IC50s approx. 1-2 µM. Piperonyl butoxide and S-methoprene were more potent inhibitors of 3HCP55940 binding IC50s: 8.2µM and 16.4µM respectively than of 3HSR141716A binding IC50s: 21µM and 63µM respectively. Binding experiments demonstrated selectivity towards brain CB1 versus spleen CB2 receptor. Benzophenanthridines reduced Kd of 3HCP55940 binding to brain membranes whereas S-methoprene and piperonyl butoxide lowered Bmax. These study compounds reduced association of 3HCP55940 and 3HSR141716A, benzophenanthridines were consistently more effective. In presence of a saturating concentration of SR141716A, S-methoprene and piperonyl butoxide increased dissociation of 3HSR141716A above that observed with SR141716A alone. All compounds activated 3HSR141716A dissociation when assayed alone, but S-methoprene was the least effective. In separate studies, phthalate diesters reduced the Bmax of 3HSR141716A without affecting Kd, increased 3HSR141716A dissociation above saturating concentration of AM251. Benzophenanthridines antagonized CP55940-stimulated and basal binding of 35SGTPyS to the G-protein of mouse brain, whereas piperonyl butoxide and S-methoprene inhibited CP55940-stimulated 35SGTPyS binding only. Inhibition of CP55940-stimulated binding of 35SGTPyS was also demonstrated with phthalates. 4-Aminopyridine-4-AP-induced release of L-glutamate from mouse brain synaptosomes was partially inhibited by WIN55212-2. The inhibitory effect of WIN55212-2 was completely neutralized by AM251, S-methoprene, piperonyl butoxide and phthalate diesters, whereas in the presence of WIN55212-2, the benzophenanthridines enhanced 4-AP-induced L-glutamate release above caused by 4-AP alone. The 3HCP55940 and 3HSR141716A binding data suggest that study compounds modify radioligand binding allosterically. The 35SGTPyS binding results suggest that chelerythrine and sanguinarine are inverse agonists of G-protein-coupled CB1 receptors, piperonyl butoxide, S-methoprene and phthalate diesters are neutral lower potency antagonists. Modulation 4-AP-evoked L-glutamate release from synaptosomes by the study compounds with WIN-55212-2 present strongly supports this latter profiling. Although these compounds exhibit lower potencies versus many conventional CB1 receptor inhibitors, further studies are warranted, given their potential to 1 modify CB1 receptor-dependent behavioral/physiological outcomes in the whole animal, and 2 serve as starting structures for synthesis of novel/more potent G-protein-coupled CB1 receptor blocking drugs.